CZ287775B6 - Method of improving organoleptic properties of meat of uncastrated males of domestic animals - Google Patents

Abstract

Uncastrated males of cattle, sheep and pig containing androgenic and non-androgenic steroids enabling development of the animal male physiognomy are subjected to fattening. Prior or during fattening, anti-LHRH vaccine for inducing primary immune response of low intensity and without significant or even measurable activity on gonadal steroidal secretion is administered to the animals in order to enable development of their male physiognomy, whereupon the anti-LHRH vaccine for inducing anti-LHRH immuno-neutralization for cancellation of the androgenic and non-androgenic steroid activity is administered to animals just prior slaughter.

Description

A method for improving the organoleptic properties of meat from uncastrated male domestic animals

Technical field

The invention relates to a method for improving organoleptic properties, in particular odor, taste and brittleness, meat of non-castrated male domestic animals, in particular cattle, sheep and pigs.

BACKGROUND OF THE INVENTION

The benefits of fattening intact instead of castrated males in the cultivation of domestic animals for meat are emphasized by specialist animals for decades. These benefits are at a higher growth rate, especially in cattle and sheep, in better use of feed rations and in the quality of the slaughtered animal meat, which are lighter but at the same time has a higher proportion of muscle tissue in all domestic animals (SC SEIDEMAN et al. of Animal Acience, 1982, 55 (4) 826-840, and M. BONNEAU, INProd. Amin., 1988, 1 (2) 133-140).

The main disadvantages of such intact male cultivation, as highlighted in the above reviews, are the unpleasant smell and taste of the meat from male pigs and sheep and the less tenderness of meat from intact male cattle and sheep, and therefore affect current surgical castration practices.

Indeed, androgenic steroids, including androstenediol, androstenedione and testosterone, which are decisive factors in gaining benefits in domestic animal husbandry in terms of rapid growth and better utilization of feed rations, are also responsible for the less fragility of meat obtained from intact male cattle and sheep. Non-androgenic steroids and / or 16-androsterone derivatives including 5α-androstene (5α-androst-16-en-3-one) are partly responsible in male pigs for the unpleasant odor and taste of meat obtained after the male has reached puberty, and these factors reduce quality meat and they are to prevent its marketing in a fresh state.

Skatol, a tryptophan-derived compound and produced by the microbial flora present in the viscera, is partly responsible for the unpleasant odor and taste of meat obtained from 35 intact male pigs. Its origin depends on ecological, nutritional and growing factors. The accumulation of skatol in adipose tissue is higher in boars and is believed to be associated with secretion of sex steroids by gonads.

The possibility of reducing or eliminating male properties in young 40 animals and secreting testicular hormones, in particular testicular steroids, through active or passive immunoneutralization against these hormones or against the hormones involved in their secretion, such as luteinizing hormone or LH and the gonadoliberin hormone ( GnRH), also known as luteinizing hormone releasing hormone (LHRH). Pigs have also been attempted to reduce the level of 5α-androstenone, a hormone of the 16-androsten group, 45 in tissue by active immunization against this substance (ED WILLAMSON et al., Livestock Production Science, 1985, 12, 251-264) or passive immunization against the same substance (R. CLAUS, Immunization with Hormenes in Reproduction Research, ed. Nieschlag, 1975). It is possible to attempt to stop or reduce testicular steroid secretion by immunoneutralization of species-specific genodotropic hormone LH (RE FALVO et al., J. Amin. 50 Acience, 1986, 63, 986-994), or by anti-LHRH immunoneutralization endogenous LHRH. Only active immunization with anti-LHRH has been recommended by various authors. In pigs, α-androstenone was reduced by this method (A. CARATY and M. BONNEAU, CR Acad. Sci. Paris 1986, 303, Serie III (16) 673-676; RE FALVO et al., J. Amin. Sci. 1986, 63, 986-994).

-1 CZ 287775 B6

In sheep, B.D.SCHANBACHER (Am. J. Physiol., 1982, 242, E201-E205) recommends anti-LHRH immunization to slow testicular development and induce castration effect in male lambs. P.S. ROBERTSON (Vet. Rec., 1979, 105, 516-517) describes immunological castration of cattle based on anti-LHRH.

Anti-LHRH-based immunoneutralization testing in laboratory animals (ARIMURA et al., Endocrinology, 1973, 93, 1092-1103; FRASER, HM et al., J. Endocr. 1974, 63, 399 ^ 106; MAKINO T. et al. , Contraception, 1973, 8 (2), 133-145; CARELLIC., And others, Proc. Natl., Acad. Sci., USA, 1982, 79, 5392-5395) and on certain domestic animals (JEFFCOATE and O., Theriogenology, 1978, 10 (4), 323-335; ROBERTSON IS et al., Veterinary Record, 1979, 105, 556; SCHANBACHER BD Am. J. Physiol., 1982,242, E201-E205), that it is possible to arrest testosterone secretion, decrease the weight of the testes and their adjacent glands, arrest spermatogenesis and, at the behavioral level, the disappearance of libido.

The results of this work prompted a return to the first immunoneutralization method, mainly based on anti-LHRH, in order to avoid traditional surgical castration for cultivation purposes.

Thus, US Patent 4,556,555 discloses a method for passive immunization of animals prior to puberty using an antiserum that contains antibodies against genadotropin.

International Patent Application WO 90/11298 discloses a method of anti-LHRH immunization at birth; LHRH sequences, coupled in tandem, coupled to a carrier protein are used to improve meat quality in pigs.

International patent application WO 88/00 056 discloses a method of immunological castration based on anti-LHRH in order to improve the social and sexual behavior of males and thus eliminate the need for surgical castration that affects their growth. The bulls were vaccinated at the age of 8 to 40 weeks and then received several booster doses.

The anti-LHRH-based vaccine is administered under the trade name VAXTRATE by Australian company WEBSTERS and is used in cows.

RE. FALVO et al. (J. Amin. Sci. 1986, 63: (986-994) immunized several groups of bulls with LHRH conjugates with human serum globulin in complete Freund's adjuvant or with muramylpeptide as adjuvant After vaccination and using several booster doses ) the authors observed high titers of anti-LHRH antibodies, but had to re-use boosters to maintain this high titer of antibodies.

I. S. ROBERTSON describes a method of immunization with LHRH bound to tetanus anatoxin or thyroglobulin and considers that this immunological procedure would allow late castration with a beneficial impact on weight gain. However, it concludes that it is necessary to further investigate procedures that would lead to a castration method applicable in practice, both in terms of the method itself and the adjuvant, since Freud's adjuvant is prohibited in practice.

Finally, A. CARATY and M. BONNEAU (C.R. Acad. Sc., Paris, Vol. 303, Serie III, No. 16, 1986) performed anti-LHRH immunization with male pigs. The authors conclude that blocking steroid production 2 to 3 weeks before slaughter would provide the opportunity to exploit the great potential of this type of animal for meat production while at the same time eliminating the problems associated with androsterone accumulation in andipose tissue. However, they conclude that further immunization techniques will have to be advanced before a method of active anti-LHRH immunization can be devised as a technique applicable to cultivation purposes.

In addition, immunoneutralization leads to serious problems in practice in terms of the safety of the intervention, particularly with regard to local reactions caused by vaccines, in particular oily vaccines, and to the risk that the meat of the animal will be unusable or of lower quality.

-2GB 287775 Β6

Proposals to improve the organoleptic properties of bovine and sheep meat have not yet been submitted.

The Applicant has now developed an industrially applicable process that allows to improve the organoleptic properties of the meat of uncastrated male domestic animals.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a method for improving the organoleptic properties of meat from non-castrated male domestic animals by feeding non-castrated male cattle, sheep and pigs containing androgenic and non-adrogenic steroids, allowing the development of the male nature of the animals.

SUMMARY OF THE INVENTION According to the present invention, an anti-LHRH vaccine is administered to animals prior to or during recovery to induce a low-intensity primary immune response without significant or even measurable effect on gonadal steroid secretion to allow the development of the male nature of the animals. shortly before slaughter, the animals are given an anti-LHRH vaccine to induce anti-LHRH immunoneutralization to reverse the effect of androgenic and androgenic steroids.

In the method of the invention, the first administered anti-LHRH vaccine is administered prior to fattening.

The first administered anti-LHRH vaccine is administered as an emulsion vaccine.

The anti-LHRH vaccine, administered prior to slaughter, is administered to pigs prior to slaughter with an aqueous type adjuvant.

Preferably, a pre-slaughter vaccine is administered containing an aluminum hydroxide gel or saponin or a mixture thereof as an adjuvant of the aqueous type.

Preferably, the vaccine is administered in an aqueous adjuvant 15 to 21 days prior to slaughter.

The anti-LHRH vaccine is given to bulls and rams prior to slaughter with an adjuvant in emulsion form.

The vaccine administered prior to slaughter is administered in emulsion form 1 to 2 months prior to slaughter.

Preferably, the pre-slaughter vaccine, in emulsion form, is administered 4 weeks to several months after administration of the first vaccine administered before or during fattening.

It is preferred to administer the vaccine in emulsion form, wherein the emulsion is of the water-in-oil type.

In one embodiment of the method of the invention, the vaccine is administered in a water-in-oil emulsion form, wherein the emulsion comprises a mixture of high purity mineral oils and nonionic surfactants.

In the method of the invention, anti-LHRH vaccines comprising an LHRH conjugate coupled to an immunogenic carrier protein selected from the group consisting of bovine serum albumin, human serum albumin, thyroglobulin, ovalbumin, anatoxin, tetanus anotoxin, equine globulins and human globulins are administered.

Preferably, the vaccine is administered transcutaneously at several sites using a needleless injection device under pressure.

Preferably a vaccine is administered comprising a conjugate that is composed of LHRH linked to an immunogenic carrier protein selected from the group consisting of equine alpha-globulin, equine alphaglobulin fraction IV-1, equine alpha-glogulin fraction IV-4, and mixtures thereof.

-3GB 287775 B6

In another method, a vaccine comprising an LHRH peptide and an immunogenic carrier protein that is linked to a carbodiimide is administered.

Preferably, a vaccine is administered comprising a conjugate composed of LHRH (3-10) which is linked to an immunogenic carrier protein selected from the group consisting of ovalbumin, equine alpha-globulin, equine alpha-globulin fraction IV-1, equine alpha- globulin of fraction IV-4 and mixtures thereof.

Alternatively, a vaccine comprising an LHRH (3-10) peptide and an immunogenic carrier protein coupled to a carbodiimide is preferably administered.

Preferably, the vaccine is administered transcutaneously at several sites using a needleless injection device under pressure.

In a first, preferred embodiment of the method, the anti-LHRH vaccine is administered to the animal preferably in the form of an emulsion, preferably during or before the fattening phase, and then shortly before the animal is slaughtered, the anti-LHRH vaccine is re-administered. The procedure can be performed in two separate administrations or by a controlled release method.

In pigs it is particularly advantageous to administer the anti-LHRH vaccine together with an aqueous adjuvant, in particular an aluminum hydroxide gel and / or a saponin. Administration is best carried out 15 to 21 days before slaughter.

Conversely, in cattle and, where appropriate, in sheep, administration is preferably carried out prior to slaughter with the vaccine in an emulsion adjuvant, preferably 1 to 2 months before slaughter. This application is best performed at least 4 weeks, and preferably several months after the first application.

In any case, the emulsion vaccine contemplated for the first application and for cattle for the second application is most preferably in the form of a water-in-oil emulsion. However, other emulsions may also be considered.

The vaccine, preferably of the emulsion type, is designed according to the invention so as to induce a primary immune response of low intensity without a measurable effect on steroid secretion by the gonads. An emulsion-based formulation is preferred, although other formulations are also useful as long as they lead to the same effect.

Pre-slaughter administration of the vaccine is performed with a vaccine formulated to cause, at this stage, arrest or significant reduction of steroid secretion, without undesirable local or overall response, capable of causing deterioration in the appearance or quality of the meat.

It is desirable, especially in pigs, to select the following two formulations of aqueous conjugate solutions: first, a base-stable water-in-oil emulsion formulation prepared from highly purified mineral, animal or vegetable oil and nonionic surfactants to induce immune a low intensity response with no measurable effect on steroid secretion by gonads, and secondly, a formulation not emulsified with an aluminum oxide or saponin gel that triggers a rapid and intense immune response leading to sufficient production of neutralizing anti-LHRH antibodies to remove or reduce gonadal steroids and to reduce the associated transport of gastrointestinal skatol.

The emulsion, in contrast to that obtained with complete or incomplete Freund's adjuvant, is a stable emulsion allowing the preparation of a vaccine for immediate use. The inflammatory skin reaction is very weak and is localized to the sites of vaccine administration in both formulations; the reaction is manifested in external examination by well-defined pimples. Its own development is limited to the dermis. The rash disappears without leaving granulomas visible during the slaughter of the animal.

-4GB 287775 B6

In another embodiment of this method, the hyperimmune anti-LHRH serum or plasma or antiLHRH monoclonal antibody is administered to the animal several days before slaughter, particularly 5 to 15 days in advance.

Passive immunization based on anti-LHRH inducing a decrease or even arrest of secretion of androgenic and non -ndrogenic steroids was achieved by intramuscular administration of hyperimmune horse plasma. Upon bringing to a sufficient level, measured by the anti-LHRH antibody titer in the serum of the immunized animal, this immunization leads to a decrease in plasma testosterone from day 3; if maintained at the same level for the next 12 days, it is sufficient to cause a reduction in tissue androsterone to below 0.50 microgram / g at which the consumer will no longer detect the unpleasant odor and particular taste of the meat from male pigs. This passive immunization method shows that maintaining a significant decrease in testosterone content for 12 days is sufficient to reduce the testosterone concentration in the tissue below the threshold. The use of monoclonal anti-LHRH antibodies secreted by porcine hybridomas or heterohybridomas may be considered for this passive immunization.

The method of administration of these formulations is preferably transcutaneous, in particular using a needle-free injection device, using a pressurized nozzle, in particular according to patent application FR-A-2 652 257.

The method according to the invention has the great advantage that it is completely safe, in particular that it does not induce local fractions that could spoil the meat.

The inflammatory reaction of the skin remains confined to the sites of application of both formulations and manifests itself when viewed externally in the form of well-defined pimples. Its internal development remains limited to the superficial dermis. The rash disappears without leaving granulomas apparent at the time of slaughter. The inflammatory response, limited to the sites of administration, reflects tolerance to both vaccine formulations and is achieved by their transcutaneous administration using a needle-free injector.

For anti-LHRH immunization, covalent binding of a LHRH peptide or fragment thereof that is not immunogenic under economic conditions of use to an immunogenic protein, called a carrier, is required.

LHRH or GnRH, whether natural or synthetic, consists of 10 amino acids, numbered 1 to 10, from the amino terminus to the carboxyl terminus, according to the following formula:

pGlu-His-Trp-Ser-Tyr-Gly-Le-Arg-Pro-Gly.NH ₂

2344 6789 10

These symbols by convention represent: pGlu, pyroglutamic acid; His, histidine; Trp, tryptophan; Ser, serine; Tr, tyrosine; Gly, glycine; Leu, leucine; Arg, arginine; Pro, proline.

The anti-LHRH immunogenic conjugates described by various authors can be prepared with respect to hapten,

(a) with all LHRH or LHRH modified at one or more sites to achieve the desired aminothermal, carboxyterminal or intermediate conjugation,

b) with one of its peptide fragments composed of 5 to 7 amino acids, modified or otherwise modified to achieve the desired amino-terminal, carboxy-terminal or intermediate conjugation,

c) with an agonist carrying a substituted amino acid, preferably at position 6, to achieve intermediate conjugation.

-5GB 287775 B6

As regards the carrier protein, bovine serum albumin, human serum albumin thyroglobulin, ovalbumin and human or equine globulins are used.

Thus, European patent application EP-A-181 236 describes immunogenic conjugates comprising a nonapeptide or decapeptide with a sequence corresponding to the last 8 amino acids of an LHRH molecule to which a lysine or a cysteine-lysine sequence at the amino terminal end is added.

In addition, patent application WO 88/05 308 discloses conjugates prepared using fragments containing 5, 6 or 7 contiguous amino acids from a natural peptide molecule, wherein each fragment comprises an N-terminal pyroglutamic acid or a carboxyterminal glycinamide, and wherein each fragment can be added in addition, an additional amino acid or a combination of amino acids at the end attached to the immunogenic protein.

Conjugating agents fall into three main categories: activating agents, homobifunctional agents, and heterobifunctional agents. While in the activating agents the binding between the two molecules is effected by binding between the two functions already present, in the other agents the binding is effected by a hydrocarbon residue, called a ligand.

Activating agents include periodic acid, used to oxidize oligosaccharide residues to aldehydes, with which the amine group of the second molecule involved in the conjugate subsequently reacts.

Carbodiimides are activating agents widely used to bind antigens to proteins; Of these, N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride, which allows the reaction to be carried out in an aqueous medium, has certainly received the greatest use. The action of carbodiimides leads to the formation of an amide bond between the carboxyl group of one protein, activated as an intermediate O-alkyl isourea, and the amino group carried by another molecule. Their advantage is in ease of use.

Homobifunctional agents are molecules carrying two identical two identical reactive groups separated by a hydrocarbon chain. These agents include glutareldehyde, which reacts with two primary amine groups, alkyl- or arylisodithiocyanates reacting with primary amines and thiols, and bisdiazotated benzidine, which binds to aromatic tyrosine residues. Bismaleimides and bisamidinates can be named for completeness. The main disadvantage of homobifunctional reagents is the difficulty of regulating the conjugates formed, since these reagents can react with two molecules of the same nature and thus lead to the formation of oligomers or polymers.

To prevent this, chemists have developed heterobifunctional agents in which the two groups have different specificities. In general, one of these groups is an N-hydroxysuccinimide ester that under mild conditions reacts with the free amino groups of the proteins to provide both N-hydroxysuccinimide and a protein linked by covalent bonding to a coupling agent on which the other function is present. Generally, this function can react with the thiols provided by the molecule to which it binds, whether they are thiols either initially present in the molecule as cysteine residues (they can form its natural component or, in the case of peptides, can be intentionally incorporated into the molecule during synthesis), or provided by such agents as 2-iminothiolane or N- [3- (2-pyridyldithio) propanoyloxy] succinimide (SPDP) after reduction.

Of the options described above, it is best to select the entire LHRH. In this case, natural LHRH is preferred over agonists such as (D-Lys ⁶ ) -LHRH, as a result of comparing the immunogenic activity of the conjugates prepared using the two peptides.

Carbodiimide is preferred over glutaraldehyde as an agent for conjugating natural LHRH with alpha-globulin.

-6GB 287775 B6

Human or equine alpha-globulin, fractions IV-1 or IV-4, is preferred over human or bovine serum albumin.

It is desirable that the vaccines contain the same active principle, preferably an alpha-glogulin conjugate with LHRH; LHRH is preferably natural LHRH and alpha-globulin of human or equine origin is especially fractions IV-1 and / or IV-1. The conjugate is preferably obtained by adding 0.5 to 2 parts by volume of a 2.5% solution of N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in 0.9% NaCl to 1 part by volume of a mixture of alpha-globulin and LHRH. in a solution containing 2 to 20 mg of this mixture in 1 mL of 0.9% NaCl. After stirring, the mixture is left overnight and then purified by gel permeation chromatography.

With respect to the carrier protein, serum albumins, particularly bovine or human, thyroglobulin, ovalbumin, human or equine globulin, and anatoxins, in particular tetanus anatoxins, may be used.

The observed large range of immune responses of male pigs to the carboxyterminal fraction of the carbodiimide-conjugated LHRH peptide or its SPDP-conjugated (D-Lys ⁶ ) -LHRH agonist with alpha-globulin, in addition to the definition of an anti-LHRH-based immunogenic conjugate using preferably a peptide comprising the carboxyl terminus of LHRH.

Consequently, and according to a second preferred embodiment of the invention, the Applicant has found it very advantageous to use a novel peptide which comprises the last 8 amino acids of LHRH, a decapeptide of the composition

Trp - Ser - Tyr - Gly - Leu - Arg - Pro - Gly.bffib

Which has a high immunogenic activity and yet does not exhibit the hormonal activity of natural LHRH.

Thus, the novel peptide (3-10) and conjugates of the peptide comprising, coupled to one of the above-mentioned immunogenic carrier proteins, ovalbumin and equine alpha-globulin, particularly to fractions IV-1 and / or IV-4, are preferred.

In the present invention, carbodiimide is preferred over glutaraldehyde and heterobifunctional agents as an agent for conjugating the LHRH (3-10) peptide particularly with equine alpha globulin or ovalbumin.

For the preferred conjugate preparation, LHRH (3-10) and carrier protein, ovalbumin or alpha-globulin, are each dissolved at a rate of 2 to 40 mg per ml of 0.1M NaCl / Ο, ΙΜ 2- (Nmorpholino) ethanesulfonic acid buffer. 0.5-2 parts by volume of a 2.5% solution of N-ethyl-.beta.-O-dimethylaminopropyl] carbodiimide in the same buffer is then added. The pH is adjusted with 1N NaOH. After stirring, the mixture was removed overnight and then purified by gel permeation chromatography to remove unbound LHRH (3-10), carbodiimide residue, and hydrolysis products.

The invention will now be described in more detail by way of examples of comparative assays of various products and vaccination methods of the invention, on the one hand, and on the other hand, assays that have demonstrated a large immune response in male pigs to the carboxyterminal portion of LHRH peptide and LHRH-based vaccination assays. in male pigs according to the invention.

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DETAILED DESCRIPTION OF THE INVENTION

I - APPLICATION OF WHOLE LHRH

A. Greater immunogenic activity of the conjugate retaining the longest carboxyterminal portion of the LHRH peptide and selecting a conjugate based on natural LHRH preferably over the (D-Lys ⁶ ) -LHRH agonist-based conjugate.

AI. - Anti-LHRH immunization of intact male pigs in male rats.

Comparison of the activity of two anti-LHRH vaccines, composed of the conjugates of the natural form LHRH (B1 and B2) or (D-Lys ⁶ ) -LHRH (Ala A2) with human albumin, the conjugates being obtained by aqueous phase carbodiimide respectively. by SDP, introduced into an oil-in-water emulsion and administered to intemuscularly to pigs and subcutaneously to rats, leads to the following conclusions:

higher natural vaccine activity based on natural LHRH: less LHRH peptide is required than conjugated (D-Lys ⁶ ) -LHRH peptide to obtain more immune response animals (Tables 1 and 3).

- Effect of dose, manifested in obtaining more animals giving an immune response with the same conjugate (Table 3).

AI. 1- Preparation of (D-Lys ⁶ ) -LHRH conjugates with albumin by SPDP.

The preparation of (D-Lys ⁶ ) -LHRH-albumin conjugates is carried out in three steps: preparation of {N- [3- (pyridyldithio) propanoyl] -D-Lys ⁶ } -LHRH, preparation of N- (3-mercaptopropanoyl) albumin, then custody.

{N '- [3- (2-Pyridyldithio) propanoyl] -D-Lys ⁶ } -LHRH is prepared by reacting excess SPDP with LHRH in aqueous solution (6 mol SPDP per 1 mol LHRH) and then quenching the reaction mixture at 4 ° C overnight, the product is obtained by centrifugation. This was dissolved in 8M urea and tested for the presence of 2-pyridyldithio groups.

N- (3-mercaptopropanoyl) albumin is obtained by treatment with 0.2 mmol SPDP per g of human albumin dissolved in 100 ml of phosphate buffer and after the reaction mixture is left overnight at 4 ° C and acidified to pH 6 by reduction with dithiothreitol. The product was then purified by gel chromatography. The assay of thiols and protein gives an indication of the degree of substitution.

Coupling is performed using one pyridyldithio group and 1.25 thiol groups. The pH is adjusted to 7 to 7.5 and after 1 hour the yield is determined by measuring the liberated 2-pyridinethione.

The average degree of substitution is derived from the data obtained. Finally, the conjugate is purified by chromatography and concentrated by ultrafiltration.

AI.2 - Preparation of LHRH-albumin conjugate using carbodiimide.

1000 mg of N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride, dissolved immediately before use in 40 ml of 0.9% NaCl, is added to 300 mg of LHRH and 300 mg of human albumin dissolved in 30 ml of 0, 9% NaCl. After stirring, the mixture was removed overnight at rt

-8EN 287775 B6 protect from light. Then the mixture is chromatographed on Sephadex G-50; the fractions corresponding to the conjugate are collected, optionally concentrated and frozen.

In the fractions containing unbound LHRH, the amount thereof and thus the average level of conjugation are determined. This value is reproducible and varies between 8 and 10 mg LHRH per 100 mg albumin.

The yield of conjugate chromatography and thus the amount (or concentration) of LHRH are derived from the ultraviolet spectra of the conjugate before and after chromatography.

A1.3 - Test procedures

The antibody titer is determined by the technique described by JEFFCOAT et al., Acta Endocr., Copenh., 1974, 75, 625-635.

Testosterone is determined directly in plasma by the RIA technique using testosterone C19-carboxymethyl ether- [125I] histamine as a radioligand.

Binding to the labeled peptide is determined after substitution of various peptides with iodine-125 according to COPPOLAND et al., Endocr., 1979 104, 1504-1506 and the technique described by JEFFCOAT et al., Acta Endocr., Copenh., 1974, 75, 625-. 635.

A 1.4 - Representation

- Tests in rats • Table 1: anti-LHRH antibody response measured by iodine-125-labeled LHRH binding level • Table 2: effect of anti-LHRH immunization on plasma testosterone concentration • Dosage

Vaccine A1: 50 µg of conjugated (D-Lys ⁶ ) -LHRH,

B1: 12 pg of conjugated LHRH.

- Tests on intact male pigs • Table 3: anti-LHRH antibody response, measured by the degree of iodine-125-labeled LHRH binding.

• Dosage

Vaccine Al: 0.5 mg conjugated (D-Lys ^s) -LHRH

A2: 6 mg of conjugated (D-Lys ⁶ ) -LHRH,

B1: 0.15 mg of conjugated LHRH,

B2: 1.20 mg of conjugated LHRH,

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Table 1

ANTI-LHRH ANTIBODY ANSWER, MEASURED BY LEVEL OF LHRH BINDING,.

Iodine-125

SERIES ANTIBODY TESTING (% pl / č)

Injection Time - week Group A1 50 µg D-Lys ⁶ -LHRH / HSA / AE Group Bl 12 µg LHRH / EDC / HSA / AE1 Titre pl / no = 50% Sc 0 - - - - - - - - - - - Sc 4 0.0 15.4 <100 7.8 7.6 <100 0.0 35.3 <100 0.0 51.1 100 ALIGN! 5 4.3 91.1 1600 14.1 85.3 980 5.9 70.8 270 15.8 94.9 2100 6 0.0 64.3 120 11.3 67.9 220 11.7 96.1 2400 0.0 68.3 280 7 14.5 72.5 400 0.0 92.4 2100 0.0 64.2 200 H, 5 67.2 240 8 19.6 70.4 310 0.0 98.8 3200 9.2 68.7 2200 0.0 38.8 310

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Table 2

EFFECT OF ANTI-LHRH IMMUNIZATION ON TESTOSTERON CONCENTRATION IN PLASMA

TESTOSTERON TEST IN PLASMA KRYS (ng / ml)

Injection Time - week Checks Group Al 50 µg D-Ly with ⁶ -LHRH / PDP / AE 1 Group B1 12 Mg LHRH / EDC / HSA Sc 0 0.40 - - 0.26 - - 0.47 - - 0.00 - - Sc 4 2.25 4.16 2.51 1.05 5.83 2.38 2.34 4.43 3.63 - 4.17 0.58 5 3.80 2.99 0.00 1.76 2.33 0.00 5,05 2.85 0.00 6.67 1.54 0.00 6 2.01 3.26 0.00 4.47 0.09 0.00 4.69 1.10 0.00 1.96 - 0.00 7 2.28 1.32 0.00 1.92 0.89 0.00 1.89 1.59 0.00 1.65 2.17 0.00 8 0.97 1.75 0.00 1.91 1.48 0.00 2.71 1.91 0.00 1,22 2.33 0.00

-11 GB 287775 B6

Table 3

ANTI-LHRH ANTIBODY ANSWER, measured by the degree of binding of LHRH,

Iodine-125.

TESTING ANTI-LHRH ANTIBODIES IN SERIES (DIL 1/50)

Titre (pl / no = 50%) Time, week D-Lys-LHRH / PDP / HSA / AEl 0.5μ _Κ (Α1) D-Lys ⁶ -LHRH / PDP / HSA / AE 16 pg (A2) Al number of pigs number of pigs number of pigs 211 219 231 242 257 205 221 227 245 251 221 T0 l.inj. T3 0 0 0 0 0 0 71.8 0 0 0 150 T4 0 0 0 0 0 0 67.3 0 0 0 100 ALIGN! T5 2.inj. 0 0 0 0 0 0 53.4 0 0 0 50 T6 0 0 0 0 0 14.2 58.5 42.1 6.6 0 55 T7 0 0 0 0 0 7.5 48.5 36.7 5 0 <50 T8 0 0 0 0 0 7.7 44.1 28.8 5 0 <50 T9 0 0 0 0 0 5 37.5 22.6 5 0 <50 Time, week LHRH- (KARBO) / HS A / AE1 0.150 mg (Bl) LHRH- (KARBO) -HSA / AE 1L, 2mg (B2) Bl B2 number of pigs number of pigs number of pigs number of pigs 213 225 247 255 261 209 215 235 241 259 247 235 259 TO l.inj. T3 0 0 0 0 0 0 0 0 0 0 T4 0 0 0 0 0 0 0 0 0 0 T5 2.inj. 0 0 0 0 0 0 0 0 0 0 T6 0 0 81.2 5 25.1 48.9 56.2 78.2 31.4 90.1 200 130 990 T7 0 0 81.7 5 49.1 64.7 49.9 86.3 52.3 92.9 170 220 880 T8 0 0 80.5 5 36.5 54.1 48.9 80.7 59.3 90.5 150 140 640 T9 0 0 74.4 5 34.5 54.5 40.1 68.5 51.6 89.1 220 220 490

A2 - Comparative test of two anti-LHRV vaccines, composed of LHRH conjugate with α-globulin, prepared with carbodiimide, resp. (D-Lys ⁶ ) -LHRH conjugate with α-globulin, prepared by SPDP and applied in an oil-in-water emulsion by intramuscular (IM) or transcutaneously (ID) pigs.

A2.1 - Preparation of (D-Lys ⁶ ) -LHRH conjugate with α-globulin by SPDP.

The method described in Example 1 was used in exactly the same manner, but albumin at 20 mg / ml was replaced by α-globulin at 6 mg / ml.

The total yield of bound (D-Lys ⁶ ) -LHRH is of the order of 45 to 50%.

Moreover, it is possible to modify the degree of α-globulin substitution and thus the degree of conjugation as desired by varying the concentration of SPDP and / or α-globulin during the preparation of MP-α-globulin.

A2.2 - Preparation of LHRH conjugate with human α-globulin by carbodiimide (EDC).

The method described in Example A1 is used in exactly the same manner, but human albumin is replaced by human α-globulin. The level of conjugation is 24 to 28 mg of LHRH bound to 100 mg of human α-globulin.

-12GB 287775 B6

A2.3 - The efficacy of the LHRH-α-globulin conjugate vaccine is higher than that of the second vaccine. Efficacy is expressed in the number of animals showing complete clearance of testosterone from plasma.

Table 4

LHRH-α-globulin. ECD (L 2 mg) IM + ID (D-Lys ⁶ ) -LHRH-α-globulin. SPDP (6mg) IM + ID Testosterone disappearance 5/10 2/10

A.3 - Wide range of immune responses of male pigs to the administration of carbodiimide conjugated LHRH peptide fraction or its agonist (D-Lys j-LHRH, conjugated by SPDP to human α-globulin).

This response is determined by comparing the percent binding of anti-LHRH and anti- (D-Lys ⁶ ) -LHRH sera by two labeled LHRH fragments, namely LHRH (3-10) with an amino-terminal deletion with LHRH (1-10), which is in the form of the free acid due to a deletion made in its carboxyterminal portion. These two fractions specifically recognize antibodies against carboxyterminal and carboxyterminal, respectively. the amino terminal portion of the peptide chain.

The extensive response to the carboxyterminal portion of the peptide chain is reflected in the number of animals having antibodies binding only the LHRH peptide (3-10) compared to those having antibodies binding exclusively LHRH in the free acid form (10/58 for anti-LHRH serum) and 3/10 for anti- (D-Lys ⁶ ) -LHRH serum).

No serum showed 100% free acid binding of LHRH, indicating specific recognition of the amino terminal fraction.

The most common were mixed responses that showed better recognition of the amino-terminal fraction of anti- (D-Lys ⁶ ) -LHRH serum than anti-LHRH serum. Of the anti-LHRH sera, only 3 out of 58 showed a cognitive ability for the aminothermal fraction of greater than 40%, while for anti- (D-Lys ⁶ ) LHRH sera this value was 4 out of 10.

B - greater immunogenicity activity of LHRH conjugate prepared with carbodiimide compared to glutaraldehyde conjugate.

B.l - Preparation of LHRH-α-globulin conjugate using glutaraldehyde.

A volume of 2.5 ml of glutaraldehyde solution containing 10 mg / ml is added dropwise over 30 minutes to 10 mg of LHRH and 50 mg of human α-globulin (Serva) dissolved in 5 ml of 0.1 M phosphate buffer at pH 7, 5; mix after each addition. The reaction mixture was left at room temperature for 2.5 h and quenched by the addition of 25 mg of sodium bisulfide dissolved in 5 mL of water. The conjugate is dialyzed at 4 ° C against 150 mM NaCl in 10 mM phosphate buffer pH 7.5 and then concentrated by ultrafiltration.

B2. - Comparative test of anti-LHRH vaccines containing the same amount of conjugated LHRH in pigs (Table 7)

- 13GB 287775 B6

Table 7

LHRH-α-globulin (optionally with carbodiimide) administered IM or ID LHRH-α-globulin (optionally with glutaraldehyde) administered IM or ID Testosterone clearance from plasma 5/10 0/10

C- Higher immunogenic activity of human α-globulin-based conjugate compared to human serumalbumin-based conjugate.

Efficacy is expressed by the number of animals showing complete clearance of testosterone from their plasma (Table 8).

Table 8

TESTS ON PIGS - INTRAMUSCULAR INJECTIONS

LHRH-HSA prepared with carbodiimide LHRH-α-globulin prepared with carbodiimide Testosterone clearance from plasma 0/5 3/5

D - Immunogenic activity of the conjugate using equine α-globulin, fraction IV-1, equivalent to the conjugate based on human α-globulin.

D.l - Preparation of LHRH conjugate with equine α-globulin by carbodiimide.

The method is used in exactly the same manner as described in Example A1, except that human albumin is replaced by equine α-globulin (Fraction IV-1).

D.2 - Subcutaneous administration of 12 gg of LHRH, conjugated to human or equine α-globulin, to rats twice at 4 week intervals.

Table 9

TESTS ON RATES

LHRH-human α-globulin fraction IV-1 LHRH-equine α-globulin fraction IV-1 Testosterone clearance from plasma 12/12 12/12

E - Greater adjuvant activity of the oil-in-water emulsion of the invention over other emulsions (Table 10).

Tests performed on pigs using the same conjugate consisting of LHRH and human α-globulin and prepared with carbodiimide, applied the same dose transcutaneously at 5 sites.

- 14GB 287775 B6

Examined emulsions: liquid oil-in-water emulsion (B), emulsion of the invention (formula C in the table), commercial emulsion, diluted with antigen (E), and oil phase emulsified with conjugate (F).

For all of these formulations, the final amount of antigen at the dose was the same.

The emulsions were prepared under conventional conditions used by those dealing with formulations of this type.

Table 10

Emulsion (B) C E F disappearance of testosterone from plasma 2/5 4/4 1/5 3/5 number of animals showing tissue concentrations of aldosterone below 0.5 gg / g 2/5 4/4 3/5 3/5

F. - The efficacy of passive anti-LHRH immunization to improve the organoleptic properties of meat, as measured by a decrease in androsterone in the tissue.

Table 11

ANDROSTENONE CONTENT IN ADIPOSE TISSUE OF CONTROL ANIMALS AND ANIMALS SUBMITTED BY PASSIVE IMMUNONEUTRALIZATION USING HYPERIMUNITY HORSE ANTI- (D-Lys ⁶ ) -LHRH PLASMA, USED IN VOLUME 9. and 13. ml.

DAY BEFORE SLAUGHTER

Checks Treated experimental animals Number of animals showing androstenone concentration below 0.50 gg / g adipose tissue 2/5 5/5

(significant difference at risk a = 0.2)

G- Efficiency and Tolerance of α-globulin-conjugated LHRH formulations using carbodiimide in the form of an oil-in-water emulsion (1st vaccine) and aluminum hydroxide and saponin (2nd vaccine); administered transcutaneously containing the same amount of LHRH at a dose at the beginning of the fattening period, respectively. 18 to 21 days prior to slaughter, using a needle-free injector known as Pigjet.

Two tests were performed at two different intervals, for Groups 1, 3 and 5 in the first interval and for Groups 2 and 4 in the second interval (Tables 12 and 13).

G.l - The efficacy of anti-LHRH immunoneutralization increases with the same vaccine volume with the number of transcutaneous sites.

-15GB 287775 B6

Table 12

Group 1 2 3 4 5 1. vaccine 1 ml (5 places) 1 ml (3 places) 1 ml (5 places) 0.4 ml (10 places) 0.4 ml (2 spots) 2. vaccine 1 ml (5 places) 1 ml (5 places) 0.4 ml (2 spots) 0.4 ml (10 places) 0.4 ml (2 spots) Testosterone disappearance or significant decrease (number of animals) 10/12 10/11 9/12 11/11 8/11 Androstenone concentration in the tissue below 0.5 pg / g (number of animals) 11/12 n 10/23 n n

n = set

G. 2 - Tolerance to vaccines is judged by the development of an inflammatory response to the skin, graded from 0 to 4, in a given animal according to the size of the pimples occurring after application; a pimple appears at every application site. The sum of the scores in each group is summarized as follows: the mean score at the end of the first week after application (App 1) and the mean slaughter score for each vaccine (S1) (Table 13). The best tolerance observed with vaccines in Group 4.

Table 13

TOLERANCES FOR TRANS-SCANIC APPLICATION DURING TWO TESTS (Test 1 with Groups 1, 3 and 5, Test 2 with Groups 2 and 4)

Group 1 2 3 4 5 Vaccine 1 or 2 1. vak. 2nd bag. 1. vak. 2nd bag. 1. vak. 2nd bag. l.vak. 2nd bag. 1. vak. 2nd bag. Number of application locations 5 5 5 5 5 2 10 10 2 2 Ad. 1 42 11 31 11 41 16 33 11 30 10 Sl 2 4 0 0 5 3 0 0 2 0 Number of animals 12 11 12 11 11

II. USE OF PEPTIDE (3-10)

A. Techniques for measuring ahti-LHRH immune response and biological efficacy by assaying testosterone in plasma and tissue.

The anti-LHRH immune response is measured by determining the antibody titer as described by JEFFCOAT et al., Acto. Endocr. (Copenh.), 19 / 4.75, 625-635.

Binding to labeled peptides is determined after substitution of various peptides with iodine-125 according to COPPOLAND et al., Endocrinology, 1979, 104, 1504-1506. Serum determination for the content of these peptides was performed by JOFFCOATA et al., As described above.

Biological efficacy is assessed by decreased or disappearance of testosterone from plasma and tissues. Plasma testosterone assay is performed directly with the plasma RIA technique using a radioligand, which is testosterone C19-carboxymethyl ether [ ¹²⁵ I] histamine (FURUYAMA S. et al., Steroids, 1972, 16, 415). The testosterone tissue test is performed on an adipose tissue sample using an RIA technique using a radioligand that is 5α- [ ³ H] androstenone, described by CLAUSEN, CR

Acad. Sci., Paris, 1974, 229-302.

- 16GB 287775 B6

B. Large scale immune response of male pigs to the carboxyterminal fraction of α-globulin conjugated to α-globulin by carbodiimide or its agonist (D-Lys ⁶ ) -LHRH conjugated to SPDP.

Bl. Preparation of LHRH-human α-globulin conjugate with carbodiimide

The conjugate is preferably obtained by adding 0.5 to 2 parts by volume of a 2.5% solution of N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in 0.9% NaCl to one volume of the α-globulin / LHRH mixture. in a solution containing 2 to 20 mg of this mixture in 1 ml of 0.9% NaCl. After stirring, the mixture is allowed to stand overnight and then purified by gel permeation chromatography.

B2. Preparation of (D-Lys ⁶ ) -LHRH conjugates with human α-globulin by SPDP.

Preparation of (D-Lys ⁶ ) -LHRH conjugates with human α-globulin is carried out in three steps, which are the preparation of {N- [3- (2-pyridyldithio) propanoyl] -D-Lys ⁶ } -LHRH, preparation of N- (3- 3-mercaptopropanoyl) of the human α-globulin derivative and finally, after one night at 4 ° C, by centrifugation of the product obtained, which is dissolved in 8M urea and the 2-pyridyldithio group is determined.

{N '- [3- (2-Pyridyldithio) propanol] -D-Lys ⁶ } -LHRH was prepared by reacting excess SPDP with LHRH in aqueous solution (6 mol SPDP per mol (D-Lys ⁶ ) -LHRH), mixture The product was dissolved in 8M urea and then assayed for 2-pyridyldithio groups.

The N- (3-mercapropropanoyl) derivative of human α-globulin is obtained by treatment with 0.2 mmol of SPDP per 0.6 g of human α-globulin dissolved in 100 ml of 0.1 M phosphate buffer; after reaction at 4 ° C overnight and acidification to pH 6, the product is obtained by reduction with dithiothreitol. The product is then purified by gel chromatography. The average degree of substitution is judged by the determination of thiol groups and protein content.

Coupling is performed using one 2-pyridyldithio group to 1.25 thiol groups. The pH is adjusted to 7 to 7.5 and then, after one hour, the yield is determined by measuring the liberated 2-pyridinethione released.

The average degree of substitution is deduced from this value. The conjugate is finally purified by chromatography and concentrated by ultrafiltration. The total yield of bound (D-Lys ⁶ ) -LHRH is of the order of 45 to 50%.

B3. The extent of the immune response of male pigs to the application of the carboxyterminal fraction of LHRH peptide conjugated under the conditions described in A1 and A2 is determined by comparing the binding capacity of anti-LHRH serum and anti- (D-Lys ⁶ ) -LHRH serum for the two labeled LHRH fragments. (LHRH (3-10) (LHRH with amino-terminal deletion) and LHRH (1-10) in free acid form (LHRH with carboxyl-terminal amino acid deletion). These two fractions recognize with greater specificity of the anti-carboxy-terminal fraction. on the one hand, and the amino-terminal fraction on the other.

In general, all animals immunized with one or the other conjugate (68/68) give a response to the carboxyterminal fraction of the peptide. Sera 3 of 10 animals out of 10 immunized with (D-Lys ⁶ ) -LHRH conjugate and sera of 10 animals out of 68 immunized with conjugated LHRH bound only the carboxyterminal fraction. Other animals give a mixed response predominantly to the carboxyterminal fraction.

-17CL 287775 B6

The response to the aminothermal fraction has no general incidence (55/68). No serum specifically bound LHRH in the free acid form.

C. Active anti-LHRH immunoneutralization assays using the LHRH conjugate (3-10) with the α-globulin IV-4 fraction and the LHRH conjugate (3-10) with the carbodiimide prepared ovalbumin.

Cl. - Preparation of LHRH (3-10) conjugate with fraction IV-4 of equine α-globulin by carbodiimide.

LHRH (3-10) of 85 mg and 170 mg of the α-globulin IV-4 fraction of IV-4 is dissolved in 12.8 ml of 0.1 M NaCl / Ο, ΙΜ 2- (N-morpholino) ethanesulfonic acid buffer. Then 212 mg of N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide dissolved in 17 ml of said solution are added. The pH is immediately adjusted to 6.0 with 1.3 ml of 1N sodium hydroxide.

The mixture is stirred and allowed to stand for 16 h at room temperature, and the conjugate is then purified by gel permeation chromatography to separate the conjugate from unconjugated LHRH. By subtracting unconjugated LHRH, the amount of bound LHRH can be adjusted. Thus, the yield of the bond can be determined.

C2. Preparation of LHRH (3-10) conjugate with ovalbumin using carbodiimide.

LHRH (3-10) in an amount of 60 mg and 120 mg of ovalbumin was dissolved in 9 ml of 0.1 M NaCl / 0.1 M 2- (N-morpholino) ethanesulfonic acid buffer. N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride in an amount of 150 mg and dissolved in 12 ml of the same buffer is then added to the solution. The pH is adjusted to 7.0 with 1N sodium hydroxide (about 1.9 ml). The mixture is left overnight at room temperature and then clarified by centrifugation. The supernatant is chromatographed on Sephadex to remove the conjugate from unreacted LHRH and carbodiimide breakdown products. LHRH binding yield (3-10) can be determined by determining the amount of unbound LHRH (3-10).

C3. - Immune response, biological efficacy and tolerance to the anti-LHRH vaccine, based on the conjugation of LHRH fragments (3-10) and fraction IV-4 of horse α-globulin by carbodiimide.

A formulation consisting of conjugated LHRH (3-10) in a water-in-oil emulsion (1st vaccine) and an aluminum hydroxide-saponin gel emulsion (2nd vaccine) was administered transcutaneously to 6 male pigs in a volume of 0.4 ml, one dose, at the beginning of the fattening period and 17 days before slaughter using a needle-free injector, known as the Pidjet injector, by dividing the dose into two 0.2 ml portions injected at 5 sites.

The immune response was maximal 10 days after the 2nd vaccine administration. The individual antibody titers (reciprocal of the dilution at which 50% iodine-125 was bound) were as follows:

50 Den 10 280 660 2700 3200 4600 1300 Day 16 290 400 2000 2400 3100 8600

-18GB 287775 B6

The biological efficacy of this immune response is manifested by the disappearance of testosterone from the plasma

On day 10 after the second vaccine was administered in all 6 animals. The disappearance of testosterone is accompanied by the disappearance of androstenone from the tissue under the same conditions.

Tolerance to the vaccine is judged by the development of an inflammatory skin reaction, graded according to the size of the pimples that appear at each site of the vaccine after administration. Local inflammation disappeared completely from day 10 after the second vaccine was administered.

Claims (18)

PATENT CLAIMS A method for improving the organoleptic characteristics of meat of non-castrated male animals, comprising feeding to non-castrated male cattle, sheep and pigs, containing androgenic and non-adenrogenic steroids, allowing the development of the male nature of animals, comprising administering to animals prior to or during fattening -LHRH vaccine to elicit a low-intensity primary immune response without significant or even measurable effect on gonadal steroid secretion to allow the development of the male nature of the animals, then prior to slaughter the animals are given an anti-LHRH vaccine to elicit anti-LHRH immunoneutralization to reverse the androgen non-androgenic steroids. The method of claim 1, wherein the anti-LHRH vaccine is administered prior to fattening. The method of claim 1, wherein the first administered anti-LHRH vaccine is administered as a vaccine in emulsion form. The method of claim 1, wherein the anti-LHRH vaccine administered prior to slaughter is administered to the pigs prior to slaughter with an aqueous type adjuvant. The method of claim 4, wherein the pre-slaughter vaccine is administered comprising an aluminum hydroxide gel or a saponin or a mixture thereof as an adjuvant of the aqueous type. The method of claim 4, wherein the vaccine is administered in an aqueous adjuvant 15 to 21 days prior to slaughter. The method of claim 1, wherein the anti-LHRH vaccine is administered to bulls and rams prior to slaughter with an adjuvant in emulsion form. The method of claim 7, wherein the pre-slaughtered vaccine is administered in emulsion form 1 to 2 months prior to slaughter. The method of claim 7, wherein the pre-slaughtered vaccine is administered in emulsion form 4 weeks to several months after administration of the first vaccine administered before or during fattening. Method according to claim 3 or 7, characterized in that the vaccine is administered in emulsion form, being of the water-in-oil type. The method of claim 10, wherein the vaccine is administered in a water-in-oil emulsion form, wherein the emulsion comprises a mixture of high purity mineral oils and nonionic surfactants. -19GB 287775 B6 12. The method of claim 1, wherein the anti-LHRH vaccine is administered comprising an LHRH conjugate coupled to an immunogenic carrier protein selected from the group consisting of bovine serum albumin, human serum albumin, thyroglobulin, ovalbumin, anatoxins, tetanus anatoxin , horse globulins and human globulins. The method of claim 12, wherein the vaccine is administered transcutaneously at multiple sites using a needleless injection device under pressure. 14. The method of claim 12, comprising administering a vaccine comprising a conjugate consisting of LHRH linked to an immunogenic carrier protein selected from the group consisting of equine alpha-globulin, equine alpha-globulin fraction IV-1, equine alphaglobulin fraction IV-4 and mixtures thereof. 15. The method of claim 14, wherein a vaccine comprising an LHRH peptide and an immunogenic carrier protein that is linked to a carbodiimide is administered. Method according to claim 12, characterized in that a vaccine is administered comprising a conjugate composed of LHRH (3-10) which is linked to an immunogenic carrier protein selected from the group consisting of ovalbumin, equine alpha-globulin, equine alpha- globulin fraction IV-1, equine alpha-globulin fraction IV-4 and mixtures thereof. 17. The method of claim 16, wherein the vaccine comprises an LHRH (3-10) peptide and an immunogenic carrier protein coupled to a carbodiimide. The method of claim 1, wherein the vaccine is administered transcutaneously at multiple sites using a needleless injection device under pressure.